The progression of mammary epithelium from an ordered, hormonal and growth factor-dependent tissue to one of metastatic neoplasia involves many steps. These include a loss of growth control, evasion of apoptosis and senescence, invasion into mesenchyme and subsequent intravasation and extravasation at secondary sites. The ability to invade is a key step in this process, and invasion is largely inhibited by the normal function of adherens junctions. Normally functioning adherens junctions are dependent upon a set of protein interactions that links neighboring cells (via E-cadherin homotypic interactions) and the intracellular actin cytoskeleton (via β-catenin). The tumor antigen MUC1 is a protein that promotes the disregulation of adherens junction proteins by sequestering β-catenin from E-cadherin. This proposal is directed at understanding the functional significance of MUC1/β-catenin interactions in cellular invasion and identifying a mechanism to interrupt these interactions as a means of inhibiting cellular invasion and metastasis.
The protein components of adherens junctions are frequently disregulated in cancer progression. In many breast cancer patients, E-cadherin expression is lost and cells no longer maintain homotypic interactions. Additionally, β-catenin has special significance due to its role not only as a cellular adhesion protein, but also as a proto-oncogene. This function is due to the involvement of β-catenin not only in E-cadherin-mediated cell adhesion, but also its presence in discrete cytoplasmic and nuclear pools, functioning as a vital player in Wnt-mediated signaling and as a nuclear cofactor (Orsulic et al., 1999). In polarized epithelium, β-catenin is a vital connection between adherens junctions and the actin cytoskeleton. Under these normal cellular conditions, any excess β-catenin is degraded through a complex signaling cascade that involves the tumor suppressor APC (adenomatous polyposis coli) (Polakis, 2000). Alternatively, under transforming conditions, excess β-catenin frequently builds up in the cytoplasm of breast cancer tumors and metastases (Schroeder et al., 2003), where it interacts with proteins which compete with E-cadherin for β-catenin binding sites (Polakis, 2000; Sommers, 1996). The most well-studied of these is the interaction between β-catenin and the tcf/lef transcription factors in the nucleus, which results in the transcription of a variety of gene products including c-myc and cyclin D1 (He et al., 1998; Shtutman et al., 1999; Tetsu and McCormick, 1999). In other transformed tissues, including breast cancer, β-catenin is also found interacting with transmembrane proteins, including the erbB receptors and the tumor antigen MUC1 (Li et al., 1998; Yamamoto et al., 1997).
MUC1 is a heavily O-glycosylated protein expressed abundantly in the lactating mammary gland in addition to being overexpressed (by greater than 10 fold) in more than 90% of human breast carcinomas and metastases (Hilkens et al., 1995; Zotter et al., 1988). In the normal mammary gland, MUC1 is expressed mainly on the apical surface of glandular epithelium, while in breast cancer, MUC1 is overexpressed, underglycosylated and apical localization is lost (Hilkens et al., 1995). The cytoplasmic domain contains potential docking sites for SH2 containing proteins, as well as a variety of putative kinase recognition sites and is tyrosine-phosphorylated both in vitro and in vivo (Schroeder et al., 2001, Zrihan-Licht, 1994 #248). MUC1 binds both GSK3β and β-catenin through motifs in the cytoplasmic tail similar to those found in the APC protein. Binding of MUC1 by β-catenin results in a reduction in the binding of β-catenin to E-cadherin in ZR-75-1 breast carcinoma cells (Li et al., 1998; Yamamoto et al., 1997). This could potentially subvert E-cadherin mediated cell adhesion in epithelial cells, promoting cell migration (Li et al., 1998). In fact, reduction of MUC1 in human breast cancer cell lines (ZR-75-1S and YMB-S) through the use of anti-sense oligonucleotides results in an E-cadherin-dependent increase in cellular adhesion (Kondo et al., 1998). Additionally, the analysis of invasive human breast cancer samples showed that MUC1 and β-catenin interactions occur in primary tumors, but to an even greater extent in lymph node metastases (Schroeder et al., 2003). Studies have shown that the MUC1/β-catenin interaction is dependent upon phosphorylation of MUC1 by the both the c-src kinase (Li et al., 2001b) and Protein Kinase C delta (PKCδ) (Ren et al., 2002). Phosphorylation of MUC1 in this system by c-src or PKCδ results in a decrease in affinity for GSK3β and an increase in binding to β-catenin.
The role of Muc1 in β-catenin-induced breast cancer progression has been genetically verified in the in vivo tumor model, MMTV-Wnt-1. The Wnts are secreted glycoproteins that bind the transmembrane frizzled receptor, resulting in a signaling cascade that inactivates the mechanism for β-catenin degradation (He et al., 1998; Polakis, 2000; Shtutman et al., 1999; Tetsu and McCormick, 1999). This results in significantly higher levels of β-catenin in the cytoplasm and the stochastic formation of unifocal mammary gland tumors in MMTV-Wnt-1 transgenic mice (Tsukamoto et al., 1988). In tumors derived from MMTV-Wnt-1 mice, MUC1 and β-catenin were found to biochemically interact in a tumor specific manner that localized to the cytoplasm and cellular membrane of transformed epithelium. To determine if MUC1 was functionally important in tumor progression in this model, MTV-Wnt-1 transgenic mice were crossed onto a Muc1-null background (Schroeder et al., 2003). Removal of Muc1 from these mice resulted in an almost 50% delay in tumor onset time. In the same study, pulsing invasive breast cancer cell lines with MUC-1 cytoplasmic domain protein fragments was found to increase their invasive capacity. These fragments represented multiple protein-interaction sites and functioned similarly to transfecting the entire MUC1 cytoplasmic tail. Localization experiments determined that these peptides tracked to invading lamellopodia (invadopodia) and colocalized with β-catenin. It was suggested that the association between MUC1 and β-catenin promotes an alternate localization of β-catenin, away from adherens junctions to sites of membrane protrusions. There, the ability of β-catenin to interact with cytoskeleton-modulating proteins promotes their redistribution and promotes cellular invasion. Therefore, when MUC1 complexes with β-catenin, it promotes the novel interaction between β-catenin and invading cell margins, possibly by acting as a scaffolding protein to bring together multiple kinases with the actin cytoskeleton at sites of membrane invasion. This complex formation may not only promote the transition from hyperplasia to neoplasia in nonmetastatic disease, but also induce the dynamic changes necessary for metastatic invasion.
Recent studies have demonstrated that MUC1 is an oncogene. Both in vitro and in vivo evidence demonstrates that overexpression of MUC1 (specifically the cytoplasmic tail of MUC1) results in transformation of breast epithelium ((Li et al., 2003) and Schroeder et al., submitted to JBC). When overexpressed in the transgenic mouse (MMTV-MUC1), approximately 60% of multiparous females develop mammary tumors with a long and highly variable latency (Schroeder et al., 2004). Ninety percent of those animals forming primary mammary gland tumors also develop pulmonary metastases. Immunoprecipitation studies between MUC1 and β-catenin determined that these two proteins interact in the tumors, but not the normal mammary glands. This data indicates that MUC1 and β-catenin interactions are not limited to the published MMTV-Wnt-1 model (Schroeder et al., 2003), but also occur in a MUC1-driven model of mammary gland tumorigenesis. Importantly, the MMTV-MUC1 transgenics are metastatic, further potentially implicating this interaction in metastatic breast cancer. Finally, in vitro evidence demonstrates that transfection of rat 3Y1 fibroblasts with MUC1 also results in not only transformation, but a specific complex formation between MUC1 and β-catenin (Li et al., 2003).
The binding site for β-catenin in the MUC1 cytoplasmic domain is surrounded by binding sites for the tyrosine kinases c-src and EGFR and the serine/threonine kinase PKCδ, and interactions between MUC1 and these kinases are increased in breast cancer cell lines and tumor tissues. Furthermore, PKCδ and src-induced phosphorylation of MUC1 promotes MUC1/β-catenin binding (Li et al., 2001). When cells are provided with peptides that mimic this entire domain, MUC1 and β-catenin colocalize in invadopodia of invasive cell lines and cellular invasion increases 5-10 fold (Schroeder et al., 2003). If smaller protein fragments are provided, representing only EGFR or GSK3β binding sites, no changes in cellular invasion or β-catenin localization is observed (Schroeder et al., 2003). These data suggest that the full-length MUC1 cytoplasmic domain acts as a scaffolding protein to promote invasion, by bringing together β-catenin with cellular kinases at invadopodia.
There is a continuing need in the art to develop treatments that are effecting in treating cancer, in particular late stage and metastatic cancers.